Previous studies have shown that N-trimethyl chitosan chloride (TMC) is a potent absorption enhancer for hydrophilic and macromolecular compounds across mucosal surfaces. TMC proved to be effective in neutral and basic pH environments where the absorption enhancing ability of chitosan is severely hampered by its insolubility in these environments. The absorption enhancing characteristics of TMC polymers with different degrees of quaternisation were investigated in vitro and in situ to identify the most effective polymer in a neutral pH environment. Different degrees of quaternisation were obtained by varying the number and duration of the reaction steps in the synthesis process of TMC. The TMC polymers were characterised with 1H-NMR spectroscopy and the degrees of quaternisation were between 22.1 and 48.8%. Everted intestinal sacs (rats) were used to determine the effect of the polymers (0.0625-0.5% w/v) on the permeation of the hydrophilic model compound, [14C]mannitol, at a pH value of 7.4. A single pass intestinal perfusion method was also used to evaluate the permeation enhancing properties of the TMC polymers under the same conditions. The results obtained from both methods clearly showed a pronounced enhancement of [14C]mannitol permeation when administered with the different TMC polymers. It was shown that the permeation enhancing effects depend on the degree of quaternisation of TMC. In both models the best permeation enhancing results were obtained with the highest degree of quaternisation of TMC (48.8%) at a concentration of 0.5% w/v.